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Northeast Arctic cod, saithe and haddock are among the most important fisheries resources in Europe, largely shipped to various continental markets. The present study aimed to map the presence and distribution of larvae of parasitic nematodes in the Anisakidae family which are of socioeconomic and public health concern. Fishes were sourced from commercial catches during winter or spring in the southern Barents Sea. Samples of fish were inspected for nematodes using the UV-press method while anisakid species identification relied on sequencing of the mtDNA cox2 gene. Anisakis simplex (s.s.) was the most prevalent and abundant anisakid recorded, occurring at high infection levels in the viscera and flesh of cod and saithe, while being less abundant in haddock. Contracaecum osculatum (s.l.) larvae, not found in the fish flesh, showed moderate-to-high prevalence in saithe, haddock and cod, respectively. Most Pseudoterranova spp. larvae occurred at low-to-moderate prevalence, and low abundance, in the viscera (Pseudoterranova bulbosa) and flesh (Pseudoterranova decipiens (s.s.) and Pseudoterranova krabbei) of cod, only 2 P. decipiens (s.s.) appeared in the flesh of saithe. Body length was the single most important host-related factor to predict overall abundance of anisakid larvae in the fish species. The spatial distribution of Anisakis larvae in the fish flesh showed much higher abundances in the belly flaps than in the dorsal fillet parts. Trimming of the flesh by removing the belly flaps would reduce larval presence in the fillets of these gadid fish species by 86–91%.

Nematode parasites of the genus

In total, 1038 farmed rainbow trout, Oncorhynchus mykiss (Walbaum, 1792), were examined for the presence of nematodes between August 2015 and December 2016. The fish originated from 15 farms in wes...

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Northeast Atlantic (NEA) mackerel (Scomber scombrus, Scombridae) represents an economically important target for the Norwegian pelagic fishing industry. Despite the commercial significance of NEA mackerel, little is known about the infections with the myxosporean parasite Kudoa thyrsites (Kudoidae). The parasite may cause post-mortem myoliquefaction of the fish skeletal muscle and therefore reduce the quality of the fish product. In this study, we examined ‘soft flesh’ occurrence in commercial size groups of NEA ‘autumn mackerel’ caught between 2007 and 2020, and investigated the prevalence and density of K. thyrsites (qPCR) and how they related to the occurrence of ‘soft flesh’. The present study is the first long-term investigation of the occurrence of K. thyrsites-induced ‘soft flesh’ in NEA mackerel. After appearing stable for over a decade, the ‘soft flesh’ occurrence increased three- to six-fold in 2019 and 2020. This increase, together with the findings that ‘soft flesh’ seems primarily to affect the commercially most valuable mackerel size group (>400 g), may have important implications for the fishing industry and the fishery management. Molecular analysis (qPCR) suggests that the prevalence of K. thyrsites is substantially higher than ‘soft flesh’ occurrence. The majority (87.4%, n = 76/87) of infected mackerel did not develop ‘soft flesh’ and only individuals with high parasite density in the musculature (12.6%, n = 11/87) showed the condition. Therefore, qPCR analyses should be used for estimating the prevalence of K. thyrsites in fish. The method may also be used to assess the risk of NEA mackerel to develop post-mortem ‘soft flesh’. publishedVersion

Kudoa thyrsites is a myxosporean parasite (Cnidaria, Myxozoa) that infects the skeletal and cardiac muscle of Northeast Atlantic (NEA) mackerel (Scomber scombrus). Heavy infections are associated with post-mortem myoliquefaction of the host skeletal muscle which reduces the quality of the fish product. The biological infection characteristics of the parasite in NEA mackerel are poorly known. This study examined the distribution of K. thyrsites in various organs of NEA mackerel from the northern North Sea, and elucidates the relationship between density of infection, developmental stage and parasite distribution in the musculature, and the extent of visible flesh myoliquefaction. Quantitative polymerase chain reaction (qPCR) data showed that K. thyrsites is unevenly distributed in the somatic musculature of the fish host, with highest density in the anterior ventral muscle sections—the belly flaps. A weak positive correlation was observed between the level of myoliquefaction and the parasite density in the fish host muscle. This relationship was also reflected by the amount and distribution of parasite developmental stages seen during histological examinations. Histological findings indicate an association between the dispersion of free myxospores and the level of myoliquefaction of the fish host muscle. Visceral organs were also found infected using qPCR, although at lower densities compared to the musculature.

The parasitic nematode Hysterothylacium aduncum commonly occurs in many fish species in the Northeast (NE) Atlantic. The parasite is considered non-pathogenic to humans. During spring and summer of 2018, lots of head-on eviscerated fresh NE Arctic cod (Gadus morhua) were shipped from Norway to Spain for commercialization. At arrival, the presence of lively roundworms (i.e. H. aduncum) in the transporting boxes was noticed. Consequently, fish lots were rejected causing substantial monetary losses to all involved parties. As part of the hazard assessment process, the epidemiology of H. aduncum in cod, NE Arctic saithe (Pollachius virens) and NE Arctic haddock (Melanogrammus aeglefinus) fished off West-Finnmark, Norway, was studied. Parasites were morphologically and molecularly identified to species level. Additionally, the viability of H. aduncum was assessed by simulating the conditions that prevail during transport of fresh cod to European market. The infection values of L4 larval stage or adult H. aduncum in the digestive tract of fish were highly variable, ranging from 100% prevalence with mean abundance (range) 238 (10–1092) in cod to 27% prevalence with mean abundance (range) 1 (0–22) in haddock. Fishing season was identified as most important explanatory factor for parasite abundance. Infection levels were higher during winter and early spring, and lower during late spring. Infection peaks of H. aduncum in the three fish species seemed to coincide with heavy predation on spawning capelin (Mallotus villosus) during winter and early spring. Thorough evisceration seems to remove most of the nematodes from the fish. However, parasites may remain hidden within the head cavities where they can stay alive and active under regular transport conditions for at least 14 days. Hysterothylacium aduncum can be completely removed from the product by evisceration, beheading and rinsing the fish prior to shipping. Alternatively, thorough cleaning or completely removal of the gills and pharynx may be considered.

A survey on Anisakis simplex (sensu stricto (s.s.)) from blue whiting, Micromesistius poutassou, in the north-eastern Atlantic Ocean revealed the occurrence of high infection levels of third larval stages in visceral organs and flesh. Larvae were genetically identified with a multilocus approach as A. simplex (s.s.). Histochemical, immunohistochemical and ultrastructural observations were conducted on 30 M. poutassou specimens. Gonads, pyloric caeca and flesh harboured encapsulated larvae of A. simplex (s.s.) but no intense host reaction was encountered around the parasite in the above organs. In the liver, the most infected organ, the larvae co-occurred with the coccidian Goussia sp. Within the granuloma around the A. simplex (s.s.) larvae, two concentric layers were recognized, an inner mostly comprising electron-dense epithelioid cells and an outer layer made of less electron-dense epithelioid cells. Macrophages and macrophage aggregates (MAs) were abundant out of the granulomas, scattered in parenchyma, and inside the MAs, the presence of engulfed Goussia sp. was frequent. In liver tissue co-infected with Goussia sp. and A. simplex (s.s.), hepatocytes showed cytoplasmic rarefaction and acute cell swelling. Results suggest that the host-induced encapsulation of A. simplex (s.s.) larvae is a strategic compromise to minimize collateral tissue damage around the larval infection sites, to facilitate the survival of both parasite and host.

Few reports exist upon the occurrence and localization of zoonotic anisakid nematodes in T. sagittatus, especially in the mantle of the squid. The occurrence and site of infection of larval anisakids in 98 T. sagittatus caught West off St. Kilda, NE Atlantic Ocean, were investigated. Squids were examined for anisakids using the UV-Press method. In total, 689 nematodes were detected in the viscera and mantle. According to morphology, all the larvae (L3) were assigned to genus Anisakis. Diagnostic allozymes and mtDNA cox2 sequence analysis permitted to genetically identify all larvae as Anisakis simplex (s.s.) (N = 100). Overall prevalence (P = 81%) and mean intensity (mI = 8.6) of infection with A. simplex are provided. Most of the larvae present in the mantle cavity were embedded in the stomach wall or attached in the outer layer of the stomach and caecum (49%). Over a third of squids (37%) hosted A. simplex (s.s.) larvae in the mantle. A novel schematized representation of larvae distribution in the mantle is provided, showing where they were mostly located. According to the results obtained, the risk of anisakiasis associated with consumption of raw or undercooked T. sagittatus should be considered.

The third-stage larvae of the parasitic nematode genus Anisakis tend to encapsulate in different tissues including the musculature of fish. Host tissue penetration and degradation involve both mechanic processes and the production of proteins encoded by an array of genes. Investigating larval gene profiles during the fish infection has relevance in understanding biological traits in the parasite&rsquo, s adaptive ability to cope with the fish hosts&rsquo, defense responses. The present study aimed to investigate the gene expression levels of some proteins in L3 of A. simplex (s.s.) infecting different tissues of blue whiting Micromesistius poutassou, a common fish host of the parasite in the NE Atlantic. The following genes encoding for Anisakis spp. proteins were studied: Kunitz-type trypsin inhibitor (TI), hemoglobin (hb), glycoprotein (GP), trehalase (treh), zinc metallopeptidase 13 (nas 13), ubiquitin-protein ligase (hyd) and sideroflexin 2 (sfxn 2). Significant differences in gene transcripts (by quantitative real-time PCR, qPCR) were observed in larvae located in various tissues of the fish host, with respect to the control. ANOVA analysis showed that relative gene expression levels of the seven target genes in the larvae are linked to the infection site in the fish host. Genes encoding some of the target proteins seem to be involved in the host tissue migration and survival of the parasite in the hostile target tissues of the fish host.

Myxosporean parasites of the genus Kudoa are fish parasites of great economic importance, as some species can affect the fish fillet quality by producing macroscopic cysts or generating post mortem myoliquefaction, commonly referred to as &lsquo, soft flesh&rsquo, Kudoa mirabilis is a &lsquo, inducing species originally described based on morphology in the musculature of Trichiurus lepturus from the Indian Ocean. An integrative morphological and genetic characterization of K. mirabilis from the type host caught off the coast of Tanzania is here provided. The spores were stellate with four unequal polar capsules, showing similarities to Kudoa thyrsites. For comparative and validation purpose, K. mirabilis was compared morphologically and genetically with K. thyrsites reference isolates, including new obtained samples from the type host Thyrsites atun caught in the SE Atlantic Ocean. Morphological analyses of spores revealed key diagnostic characters clearly distinguishing the two Kudoa species. Phylogenetic analyses based on SSU and LSU rRNA genes demonstrated that K. mirabilis is a distinct and valid species, representing a sister group to a K. thyrsites subclade that comprises several isolates from Japan and one single isolate from South Africa. This finding raises questions about the true diversity likely hidden in the K. thyrsites complex.

A total of 80 stockfish fillets of Northeast Arctic cod (Gadus morhua), traditionally open-air-dried in northern Norway, was examined for the presence and viability of larval parasitic nematodes of the family Anisakidae. Anisakids (particularly those belonging to genera Anisakis and Pseudoterranova) are of public health and economic concern globally, since they are responsible for an underestimated fish-borne zoonotic disease called anisakidosis (anisakiasis when caused by members of the Anisakis genus). Stockfish fillets were inspected for anisakids by candling and artificial (pepsin) digestion methodologies. The recovered nematodes (n = 342) were morphologically identified to genus level and their viability assessed. Subsamples of anisakid larvae (n = 31) were identified by molecular/genetic markers inferred from sequences analyses and real time polymerase chain reaction (RT-PCR) of the mtDNA cox2 gene, as Anisakis simplex sensu stricto (s.s.) (n = 29) and as Pseudoterranova decipiens (s.s.) (n = 2). This is the first time a RT-PCR primer/probe system was used to identify anisakids in a processed fishery product. Anisakis simplex (s.s.) larvae were found in 81% of the fillets, with average (range) 4 (0–35). In total, 338 A. simplex (s.s.) and 4 P. decipiens (s.s.) larvae, all dead, were recovered from the fillets. Anisakids were devitalised by the air-dried stockfish production process in 7.5 months (common stockfish production time from sea to plate). The results suggest that there is a negligible risk of acquiring anisakidosis from consumption of air-dried stockfish. Further research is recommended to evaluate if anisakids can be devitalised in five months (i.e. minimum stockfish production time). The health risk for sensitized consumers posed by the potential presence of anisakid allergens in stockfish needs to be assessed. This is the first report on the viability of anisakid larvae in an unsalted, naturally dried fishery product. Drying could represent an alternative and efficient treatment for the inactivation of anisakids in fishery products. Trimming of the belly flaps of highly parasitized cod may reduce the number of anisakids in stockfish by 74%.

13 pages, 6 figures, 4 tables, Atlantic cod (Gadus morhua) is among the most important commercial fish species on the world market. Its infection by ascaridoid nematodes has long been known, Pseudoterranova even being named cod worm. In the present study, 755 individuals were sampled in the Barents, Baltic and North Seas during 2012–2014. Prevalences for Anisakis in whole fish and in fillets in the different fishing areas varied from 16 to 100% and from 12 to 90% respectively. Abundance was also greatly influenced by the sampling area. Generalized additive model results indicate higher numbers of Anisakis in the North Sea, even after the larger body size was accounted for. Numbers and prevalence of Anisakis were positively related to fish length or weight. The prevalence of parasites in whole fish and in fillets was also influenced by the season, with the spring displaying a peak for the prevalence in whole fish and, at the same time, a drop for the prevalence in fillets. Whereas 46% of cod had Anisakis larvae in their fillets, the majority (39%) had parasites mainly in the ventral part of the fillet and only 12% had parasites in their dorsal part. This observation is of importance for the processing of the fish. Indeed, the trimming of the ventral part of the cod fillet would allow the almost total elimination of ascaridoids except for cod from the Baltic Sea where there was no difference between the dorsal and the ventral part. The presence of other ascaridoid genera was also noticeable in some areas. For Pseudoterranova, the highest prevalence (45%) in whole fish was observed in the Northern North Sea, whereas the other areas had prevalences between 3 and 16%. Contracaecum was present in every commercial size cod sampled in the Baltic Sea with an intensity of up to 96 worms but no Contracaecum was isolated from the Central North Sea. Non-zoonotic Hysterothylacium was absent from the Baltic Sea but with a prevalence of 83% in the Barents and the Northern North Sea. A subsample of worms was identified with genetic-molecular tools and assigned to the species A. simplex (s.s.), A. pegreffii, P. decipiens (s.s.), P. krabbei, C. osculatum and H. aduncum. In addition to high prevalence and abundance values, the cod sampled in this study presented a diversity of ascaridoid nematodes with a majority of fish displaying a co-infection. Out of 295 whole infected fish, 269 were co-infected by at least 2 genera., The present survey was financially supported by the European Union's 7th Framework Program for Research, Technological Development and Demonstration, project Parasite risk assessment with integrated tools in EU fish production value chains (PARASITE), Grant agreement (GA) no. 312068.

This review provides an inventory of the biodiversity of the anisakid species identified so far from fish and marine mammals of the NE Atlantic Ocean. The paper reviews and discusses various taxonomical and epidemiological aspects related to biodiversity assessment, with emphasis on: (1) taxa recognized as ‘biological species’ based on molecular/genetic markers; (2) current molecular/genetic approaches to identify the species at different developmental stages; (3) ecological data related to the actual geographical distribution and definitive host preferences of the species; (4) their distribution in various, commercially important fish species in northern European waters; (5) their possible occurrence in farmed fish; and, finally, (6) an update of their zoonotic potential as causative agents of anisakidosis in humans.

The species of Anisakis constitute one of the most widespread groups of ascaridoid nematodes in the marine ecosystem. Three closely related taxa are recognised in the A. simplex (s. l.) complex, i.e. A. pegreffii, A. simplex (s. s.) and A. berlandi. They are distributed in populations of their intermediate/paratenic (fish and squids) and definitive (cetaceans) hosts. A panel of seven microsatellite loci (Anisl 05784, Anisl 08059, Anisl 00875, Anisl 07132, Anisl 00314, Anisl 10535 and Anisl 00185), were developed and validated on a total of N = 943 specimens of A. pegreffii and A. simplex (s. s.), collected in fish and cetacean hosts from allopatric areas within the range of distribution of these parasite species. In addition, the locus Anisl 7, previously detected in those Anisakis spp., was investigated. The parasites were first identified by sequence analysis of the EF1 α-1 nDNA. The panel of the microsatellites loci here developed have allowed to: (i) detect diagnostic microsatellite loci between the two species; (ii) identify specimens of the two species A. pegreffii, A. simplex (s. s.) in a multi-marker nuclear genotyping approach; (iii) discover two sex-linked loci in both Anisakis species and (iv) estimate levels of genetic differentiation at both the inter- and intra-specific level.

Myxozoans of the genus Kudoa (Myxosporea, Multivalvulida) infect marine and estuarine fish species worldwide. Some Kudoa species are of concern to the seafood industry since they may generate macroscopic cysts in the fish host's musculature, or cause post mortem myoliquefaction, commonly known as 'soft flesh'. One of the economically most important species is K. thyrsites, a myoliquefactive myxosporean parasite that occurs in many wild and cultured marine fish species worldwide. Here we identified a K. thyrsites isolate as the causative agent of myoliquefaction in silver scabbardfish Lepidopus caudatus from the Alboran Sea (western Mediterranean Sea). For comparative and validation purposes, the morphological and molecular characteristics of the isolate were compared with fresh spores of a K. thyrsites isolate infecting Atlantic mackerel Scomber scombrus from the Norwegian Sea. Myxospores of both isolates shared a stellate appearance and contained 4 unequal pyriform polar capsules (1 large, 1 small and 2 intermediate). These morphological traits were consistent with all other previously described K. thyrsites isolates. Moreover, the small subunit rDNA sequences of the Mediterranean and Norwegian Sea isolates revealed 100% similarity, and matched 100% with K. thyrsites isolates previously recorded in myoliquefactive Atlantic mackerel from the North Sea and off southern England. The findings suggest that K. thyrsites is the primary cause of myoliquefaction in silver scabbardfish from the Alboran Sea. This report represents the first morphological and molecular characterization of K. thyrsites in the Mediterranean Sea. A set of new allometric characters is proposed as additional descriptors for more accurate and specific description of kudoid myxospores.

41 pages, 1 figure., Background Nematodes of the genus Anisakis parasitize many commercial fish species and are responsible for a fish-borne zoonosis (anisakiasis) and allergic reactions. Anisakis can also cause consumer distrust in fishery products and economic losses to the fish industry. Scope and approach We review current socioeconomic, legislative, risk management and human health problems caused by the occurrence of Anisakis in fishery products and discuss possible strategies to mitigate them. Key findings and conclusions Visual inspection (and candling) of fishery products as required by EU legislation is not efficient for parasite detection. Consequently, visible (and non-visible) Anisakis reach the market and may be detected (and eaten) by consumers. Marine fish appears to be the only industrial food product that is at high risk of containing parasites when placed on the market. Anisakiasis and allergy to Anisakis are hidden, underestimated emerging zoonoses worldwide. There is a need to better understand the impact of these zoonoses on individual health and particularly exposed human populations, and to assess the risk posed by Anisakis allergens in fishery products. Quantitative risk assessment (QRA) is identified as an appropriate methodology as it estimates the risk from fishing ground to human disease. Improvements in parasite control legislation and procedures (e.g. establishment of research-based and standardized parasite detection methodologies, appropriate sampling strategies, development of non-destructive methods for detection and removal of nematodes from fish products), suitable for use by seafood businesses, are recommended to improve protection of consumers and to protect the industry by minimizing Anisakis-associated economic losses. QRA may help to provide the scientific basis for improved food safety legislation and strategies to reduce the risk of anisakiasis/allergy in humans.

The species ofAnisakisconstitute one of the most widespread groups of ascaridoid nematodes in the marine ecosystem. Three closely related taxa are recognised in theA. simplex(s. l.) complex, i.e.A. pegreffii,A. simplex(s. s.) andA. berlandi.They are distributed in populations of their intermediate/paratenic (fish and squids) and definitive (cetaceans) hosts. A panel of seven microsatellite loci (Anisl 05784,Anisl 08059,Anisl 00875,Anisl 07132, Anisl 00314, Anisl 10535andAnisl 00185), were developed and validated on a total ofN= 943 specimens ofA. pegreffiiandA. simplex(s. s.), collected in fish and cetacean hosts from allopatric areas within the range of distribution of these parasite species. In addition, the locusAnisl 7, previously detected in thoseAnisakisspp., was investigated. The parasites were first identified by sequence analysis of the EF1α-1 nDNA. The panel of the microsatellites loci here developed have allowed to: (i) detect diagnostic microsatellite loci between the two species; (ii) identify specimens of the two speciesA. pegreffii, A. simplex(s. s.) in a multi-marker nuclear genotyping approach; (iii) discover two sex-linked loci in bothAnisakisspecies and (iv) estimate levels of genetic differentiation at both the inter- and intra-specific level.

The Argentinean short-finned squid (Illex argentinus) is an oceanic, neritic species widely distributed off the east coast of South America, representing the most abundant commercially exploited squid species in these waters. Despite the great commercial importance of Argentinean short-finned squid as a food resource, and as frozen product exported to Europe, the presence of zoonotic anisakid nematodes, especially in the mantle of the squid, is poorly known. The occurrence and site of infection of larval ascaridoid nematodes in 70 I. argentinus caught off the Falkland Islands were investigated. Squids were examined using the UV-Press method. In total, 30 nematodes were detected in the viscera and mantle. According to morphology, 27 were third-stage larvae (L3) belonging to genus Anisakis, while three were L3 assigned to Hysterothylacium. Anisakis pegreffii (n = 27) were identified by sequence analysis of the mtDNA cox2 and the partial EF1 α-1 region of nDNA genes; Hysterothylacium aduncum (N = 3) were identified by sequence analysis of the ITS rDNA region. These findings represent the first molecular identification of A. pegreffii and H. aduncum in I. argentinus. Both prevalence (P = 15.7%) and abundance (A = 0.39) of infection with A. pegreffii were low, and even lower values of infection were recorded for H. aduncum (P = 2.1%, A = 0.04). Only 3 out of 70 (4.3%) squids hosted A. pegreffii larvae in the mantle. Larvae infecting viscera were coiled and mainly attached to outer surface of visceral organs. Mantle-infecting larvae were situated in the posterior half. Thus, these results suggest that – although low - the risk of acquiring anisakiasis from consumption of raw, marinated and/or undercooked short-finned squid products still exists.

A total of 4184 farmed Atlantic salmon (Salmo salar) were sampled and subsequently examined for nematodes between January 2014 and July 2015. The fish originated from 37 salmon farms along the coast of Norway and represented all salmon-producing counties. Samplings took place at processing facilities during regular slaughtering procedures and consisted of 3525 harvest quality salmon processed for human consumption and 659 discarded salmon including runts and fish discarded for other quality defects. Both viscera and musculature (fillets including belly flaps) of the salmon were screened by applying the UV-press method. No nematodes were found in any of the harvest quality salmon. The only nematode findings were from the viscera of three runts (loser fish) originating in southern or western Norwegian farms, and consisted of two Anisakis simplex (s.s.) larvae and three adults of the non-zoonotic species Hysterothylacium aduncum. The absence of nematodes in the harvest quality salmon relates most likely to the diet since healthy and normally developing salmon seem to rely exclusively on the heated and extruded dry-feed, which cannot contain any viable parasites. The runts, however, may feed opportunistically on whatever prey available in the cages, which apparently facilitates the transfer of nematodes. Thus, the present results suggest that the risk of any parasitic nematodes to occur in the flesh of farmed Norwegian salmon intended for human consumption is very low.

Capelin (Mallotus villosus) is among the most abundant fish species in the Barents Sea, and represents a critical food source for many predators in the area including Atlantic cod and harp seal. In Norway, the fish is of economic importance since whole capelin and roe are valuable export products. Despite its economic and ecological importance, the parasites of Barents Sea capelin are poorly known. However, the presence of parasites in the edible parts may adversely affect product quality and consumer safety. During the main annual catching seasons of 2009-2012, we investigated the diversity and infection dynamics of ascaridoid nematodes in capelin (n = 620) from the southern Barents Sea. Three anisakid species were identified by genetic or molecular methods; Anisakis simplex (s.s.), Contracaecum osculatum sp. B, and Hysterothylacium aduncum, with C. osculatum sp. B as the most prevalent and abundant species. The present findings suggest that the ascaridoid species composition in capelin reflects its trophic position in the Barents Sea ecosystem. There appears to be a link between infection level of the nematode species and the preferred prey organisms of the different developmental phases of capelin. Thus, the higher abundance of C. osculatum sp. B compared to A. simplex (s.s.) and H. aduncum may be related to more extensive feeding on calanoid copepods over a wider ontogenetic size range including adolescence, while the main intermediate hosts of the latter nematode species, i.e. euphausiids and amphipods, appear to be the preferred prey of larger capelin.

Ascaridoid nematodes belonging to the genera Anisakis and Pseudoterranova are heteroxenous parasites, involving marine mammals as definitive hosts in their life-cycles, whereas crustaceans (krill), fish and squids acting as intermediate/paratenic hosts. These parasites are considered among the most important biological hazards present in “seafood” products. Indeed, larval stages of the Anisakis and Pseudoterranova have been reported as etiological agents of human infections (anisakidosis). We developed a primers/probe system for the identification of five species of anisakid nematodes belonging to the genera Anisakis (i.e. A. pegreffii and A. simplex (s. s.)), and Pseudoterranova (i.e. P. decipiens (s. s.) , P. krabbei and P. bulbosa) to be used in a real time polymerase chain reaction (RT-PCR) with specific primers based on the mtDNA cox2 gene. Because those anisakid species could be also found in co-infection in some fish species with the raphidascarid nematode Hysterothylacium aduncum , a species-specific primer probe system to be used in RT-PCR for this nematode species was also developed. The detection limit and specificity of the primer/probe systems were evaluated for each of the six nematode species. Singleplex and multiplex RT-PCR protocols were defined and tested. The detection limit of the nematode species tissue was lower than 0.0006 ng/μl. Efficiency ( E ) of primers/probe systems developed was carried out by standard curve; E value varied between 2.015 and 2.11, with respect to a perfect reaction efficiency value of E = 2. Considering the sensibility and quantitative nature of the assays, the new primers/probe system may represent a useful tool for future basic and applied research that focuses on the identification of Anisakis spp., Pseudoterranova spp. and H. aduncum larvae in fish, even in co-infections, with a potential for application in fish farming, fish processing industries, fish markets, and food producers.

10 pages, 4 figures, 2 tables, Atlantic mackerel (Scomber scombrus) ranks among the most valuable fish species in Europe. The NE Atlantic mackerel population is considered to comprise three main stocks (southern, western and North Sea), with variable proportions of these three intermixing at the northerly feeding grounds. The southern and western mackerel stocks have moved over the past 4–5 years further north- and westward. Consequently, large-scale mackerel fishing and processing have become thriving industries in Iceland and the Faroe Islands in just a few years. The mackerel population structure in the Mediterranean Sea is less well known but seems to comprise at least one, more or less isolated, spawning component. Although mackerel is an important food resource, systematic and concerted epidemiological surveys of Anisakis species in Atlantic mackerel from European fishing grounds have been lacking. As part of the EU FP7 PARASITE project (GA no. 312068), occurrence and specific identity of Anisakis spp. from 1801 mackerel from Northeast Atlantic and Mediterranean waters was investigated. In general, mackerel caught at the Atlantic fishing grounds exhibited markedly higher Anisakis spp. infection levels than fish from the Mediterranean localities. Mackerel caught off NW Spain and Portugal (ICES VIIIc, IXa) showed highest overall and muscular prevalence, reaching 87% and 52%, respectively, which differed significantly from all other Atlantic samples. Lowest overall Anisakis spp. prevalence and abundance was recorded in mackerel from Faroe Isles waters, while lowest muscular infection levels were found in the samples from the North Sea. Genetic nematode species identification showed that A. simplex (sensu stricto) is the dominating species in mackerel from the Atlantic areas, while A. pegreffii dominated in the samples from the Mediterranean Sea. The latter species showed generally low prevalence and intensity in the flesh, not exceeding 6% and one larva, respectively. While A. simplex (s. s.) and A. pegreffii seem to co-occur in mackerel from off NW Spain and Portugal, several A. pegreffii were also recorded in mackerel from the North- and Norwegian Seas. These findings imply that the actual mackerel started their feeding migration in waters south to the British Isles, which include parts of the sympatric area of the two sibling species. Thus, A. pegreffii could prove a useful supplementary marker to track migration routes of the different mackerel stock components in NE Atlantic waters., The present survey was financially supported by the European Union's 7th Framework Program for Research, Technological Development and Demonstration, project Parasite risk assessment with integrated tools in EU fish production value chains (PARASITE), Grant agreement (GA) no. 312068

18 pages, 10 figures, 6 tables.-- Arne Levsen ... et al., Harvesting and exploiting limited fisheries resources in a sustainable manner also implies achieving maximum added value from the raw material. However, the presence of parasites in the products may adversely affect consumer perception and/or pose a direct health hazard. As a major stepping-stone of the PARASITE project, an epidemiological survey was carried out to provide the basis for analysis and prediction of consumer exposure risk due to the presence of anisakid nematodes in fish from European wild-catch fisheries. The project consisted of nine separate workpackages (WP) where the exposure risk assessment survey was organized within WP2. The surveillance task also provided the data or samples needed for data management and sample storage (WP3, Biobank), molecular and genetic parasite species identification (WP4), and statistical modelling and inference (WP8). In total 17,760 fish belonging to 16 teleost species were examined for anisakids, with special emphasis on economically and ecologically important species such as Atlantic mackerel, herring, European hake, Atlantic cod and anchovy. The target fish species were sampled at four major European fishing areas including the Barents Sea, North Sea, Baltic Sea, Grand Sole Bank, waters off NW Spain and Portugal, central and western parts of the Mediterranean Sea, and the Adriatic Sea. Thus, the survey represents the largest and most comprehensive epidemiological data compilation of anisakids ever generated in terms of geographical range as well as number of fish host species and sample size. An important requirement of the survey was the use of commonly accepted nematode detection methods, i.e. the UV-press method or artificial digestion, to quantify infection level and spatial distribution of anisakid larvae in the target fish species. The basic layout, set-up and principles of the method, along with a discussion of possible source of errors are described. Additionally, the molecular and genetic markers which were used to identify and characterize different species and populations of anisakids from the targeted fish host species and geographical areas, are reviewed as well. Some basic parasite infection characteristics of each fish host species, and any relationships with the presumably most important infection predictors, i.e. fish host body size and fishing locality, are presented and discussed. Emphasis is put on anisakid occurrence in the flesh of the fish. Based on the findings, a graphical exposure risk profile is introduced, including fish species or products thereof, which due to common processing or preparation practices, are at highest risk to act as source of anisakiasis in Europe, The present work was financially supported by the European Union’s 7th Framework Programme for Research, Technological Development and Demonstration, project Parasite risk assessment with integrated tools in EU fish production value chains (PARASITE), Grant Agreement (GA) no. 312068

This review addresses the biodiversity, biology, distribution, ecology, epidemiology, and consumer health significance of the so far known species of Anisakis, both in their natural hosts and in human accidental host populations, worldwide. These key aspects of the Anisakis species' biology are highlighted, since we consider them as main driving forces behind which most of the research in this field has been carried out over the past decade. From a public health perspective, the human disease caused by Anisakis species (anisakiasis) appears to be considerably underreported and underestimated in many countries or regions around the globe. Indeed, when considering the importance of marine fish species as part of the everyday diet in many coastal communities around the globe, there still exist significant knowledge gaps as to local epidemiological and ecological drivers of the transmission of Anisakis spp. to humans. We further identify some key knowledge gaps related to Anisakis species epidemiology in both natural and accidental hosts, to be filled in light of new 'omic' technologies yet to be fully developed. Moreover, we suggest that future Anisakis research takes a 'holistic' approach by integrating genetic, ecological, immunobiological, and environmental factors, thus allowing proper assessment of the epidemiology of Anisakis spp. in their natural hosts, in human populations, and in the marine ecosystem, in both space and time.

9 pages, 3 figures, 5 tables.-- Simonetta Mattiucci ... et al., The Atlantic herring is a schooling, pelagic species that inhabits both sides of the North Atlantic Ocean. Herring stock identification is usually based on several approaches, including fish meristic characters, population genetic analysis and the use of parasite species composition. A total of 654 Anisakis spp. larvae collected from herring of four fishing grounds in the Norwegian Sea, Baltic Sea, North Sea, and the English Channel off the French coast, was identified to species level using diagnostic allozymes and sequence analysis of EF1 α−1 nDNA and the mtDNA cox2 genes. Population genetic differentiation of Anisakis simplex (s. s.) among the different fishing areas was estimated, at the intraspecific level, on the basis of mtDNA cox2 sequences analysis. Spatial comparison based on molecular variance analysis and Fst values was performed for the collected specimens (among regions). Haplotype network construction showed relevant differences in haplotype frequencies between samples of A. simplex (s. s.) from the different geographical areas. Results indicate a genetic sub-structuring of A. simplex (s. s.) obtained from herring in different areas, with the population from the Norwegian Sea being the most differentiated one, and with North Sea and Baltic Sea populations being most similar. The population genetic structure of A. simplex (s. s.) was in accordance with the herring population genetic structure throughout the host’s geographical range in the NE Atlantic. Results suggest that mtDNA cox2 is a suitable genetic marker for A. simplex (s. s.) population genetic structure analysis and a valuable tool to elucidate the herring stock structure in the NE Atlantic Ocean, The research was carried out with funding received from the European Union’s Seventh Framework Programme for research, technological development and demonstration, under the grant agreement no. 312068 “PARASITE”

Parasitic anisakid nematodes commonly occur in the musculature and visceral organs of many fish species from the North Atlantic. In this respect, the presence of the third stage larvae of Anisakis spp. in the fish musculature may pose a potential consumer hazard due to the parasite's ability to cause anisakidosis. Thus, knowledge on the occurrence and distribution of these potentially zoonotic parasites in the commercially important North Atlantic fish species is crucial in order to evaluate and consequently prevent human infections. In the present study, 300 Sebastes mentella from three North Atlantic fishing grounds (Northern North Sea: Tampen; Barents Sea: off Bear Island; Irminger Sea: off SE Greenland) were examined for anisakid nematodes, with emphasis on occurrence and distribution in the musculature. Overall larval prevalence and mean intensity were significantly higher in redfish from Tampen (94%; 13.5±20.0) and Bear Island (94%; 14.5±19.4) than in fish from SE Greenland (75%; 6.0±5.8; p0.01). The same trend was observed for larval infection in the musculature showing prevalence and mean intensities of 79%, 73%, and 55%, and 5.9±6.6, 5.8±6.5, and 3.2±2.4, in the musculature of redfish from Tampen, Bear Island, and Greenland, respectively. Conventional microscopy and rDNA ITS-gene sequencing of various subsamples of muscle-dwelling nematode larvae of redfish from every catching locality revealed the presence of two anisakid species; Anisakis simplex sensu stricto and the non-zoonotic Hysterothylacium aduncum. Since the larvae of H. aduncum typically occur in or on the viscera of fish, our findings of two specimens in the belly flaps of redfish were unusual. Additionally, more than 92% of the muscle-dwelling larvae occurred in the belly flaps, i.e. the hypaxial part of the musculature surrounding the visceral organs on either fish side. Thus, trimming the fillets of beaked redfish by removal of most of the belly flaps would significantly reduce the probability of anisakid nematode larvae to be present in the final product.

Wild caught marine fish are commonly infected with anisakid nematodes lodging in the intestinal linings or in the fish muscle. One of the most commonly found nematode parasites in marine fish is Anisakis simplex. During production of mince from the muscle of wild caught Anisakis-infected fish, the larvae would be disrupted during mince production. Any bacteria within or on the surface of such larvae are during the mincing process evenly distributed throughout the mince, and could thus possibly affect the spoilage rate of the final products. To explore if or how any bacteria associated with muscle-invading Anisakis larvae may affect the spoilage rate of fish mince, a controlled storage trial was conducted. Fillets of farmed Atlantic cod (Gadus morhua), exclusively fed on dried and heat-treated compound feed and hence expectably free from Anisakis larvae, were aseptically collected and homogenised. Fish mince aliquots were added different volumes of Anisakis homogenate based on larvae which were freshly sampled from the visceral cavity of NE Atlantic blue whiting (Micromesistius poutassou). The volumes of added parasite homogenate (parasite(+)-samples) reflected different infection intensities from 15 (low) to 50 (high) larvae per 100 g fish fillet, representing an actual Anisakis intensity range in the flesh of blue whiting. The samples were kept at 4 °C for 15 days and subjected to microbiological, sensory and chemical evaluation at 3 days intervals. Upon visual examination and plate count measurements (PC) on Iron Agar Lyngby (IAL), the samples without any parasite additives (no[parasite]) spoiled differently and more rapidly than any of the parasite(+)-samples. However, H2S-producing bacteria were only recorded in the latter samples, which were also the only ones that showed increased levels of the spoilage indicator substance trimethylamine (TMA). Moreover, the parasite(+)-samples changed their sensory characteristics at a later stage compared to the no[parasite]-samples. Although some cultures of H2S-producing bacteria were found on IAL, molecular identification by PCR-DGGE of the actual bacteria was not conclusive. Psychrobacter sp. which has no or only little spoilage activity, was identified in all samples until trial day 9, but was probably outgrown by the stronger spoilers Pseudomonas fluorescence/fragi and Photobacterium phosphoreum. Thus, and somewhat unexpected, our findings indicate that – under the present trial conditions – fish mince contaminated with bacteria which originate from Anisakis larvae, spoiled less rapidly than samples without any parasite-related bacteria present. Moreover, the shelf-life of fish mince was apparently not reduced by the presence of bacteria transferred to the mince by Anisakis larvae. publishedVersion

Previous studies have shown that Grey gurnard from the North Sea is comparatively heavily infected with the larvae of the parasitic and potentially consumer hazardous nematode Anisakis simplex (s.l.). Especially the high abundance of Anisakis larvae in the fish flesh is of concern regarding the possible intensified utilisation of Grey Gurnard as a food resource. To gain better knowledge of the nematode burden with emphasis on abundance and distribution in the fish flesh, various Grey gurnards from 6 localities across the North Sea, each representing either western, northern or eastern fishing grounds, were examined for anisakid nematodes during summer of 2010 and 2012. The overall prevalence of A. simplex (s.l.) larvae in the gurnards from all localities (n = 188) was nearly total, i.e. only a single fish from the northernmost locality (Viking Bank) was apparently not infected. GLM-analyses revealed that catching locality had by far the strongest effect on larval infection probability, both in total (flesh + viscera) and in the fish flesh, while body size (weight) was a weak but still significant predictor of overall A. simplex (s.l.) abundance, but not in the flesh. Thus, the overall larval abundance in the gurnards from the two western localities off the east-coast of British mainland was significantly higher compared to the other catching localities. In the former localities, the overall mean abundance of A. simplex (s.l.) larvae was 35.0 ± 23.9 and 92.2 ± 87.4, and ranged from 14 to 114 and 9 to 397, respectively. It was further shown that 93.8% of the larvae infecting the fish flesh were situated in the belly flaps, i.e. the ventral portion of each flesh side or fillet, independent of catching locality and fish size. However, trimming of the fillets by removing the belly flaps may reduce the abundance in the fillets to, on an average, 0.3 larvae.

Many commercially utilised marine fish species are regularly infected with the larvae of parasitic nematodes of the genus Anisakis . Most of the larvae are found in the intestinal lumen of the fish, or on the organs of the visceral cavity. Some may, however, migrate from the intestine into the flesh of the fish, sometimes deeply into the fillets. The intestine of fish has a rich and diverse microbiota, including members of the specific spoilage bacteria (SSB) of seafood. Thus, the muscle invading nematodes will inevitably have a high load of bacteria in their intestinal tract and on their surfaces. While many studies are concerned with the quality reducing or human pathogenic properties these larvae represent, the present study aims to investigate the role of muscle-invading larvae of the species Anisakis simplex with respect to bacterial contamination of the flesh of post-harvest pelagic fish. Tissue samples of Anisakis -positive and Anisakis -negative body muscle, and samples of the fish intestine were collected from freshly caught blue whiting ( Micromesistius poutassou ) and processed for subsequent histological and microbiological examination. In Gram stained sections, the parasite intestine was densely populated with Gram-positive bacteria. The culture based quantitative analyses showed seven times higher numbers of colony forming units (CFU)/g for plate counts and ten times higher for SSB/g, when comparing the Anisakis -negative samples with the Anisakis -positive samples. This indicates that the parasite larvae act as vector for bacteria which contaminate the fish flesh. Molecular qualitative analyses were performed by 16S rRNA gene examinations, PCR-DGGE and sequencing. The general microbiota was determined, and some bacteria such as Shewanella sp., Photobacterium phosphoreum , and Vibrio sp., all known contributors to the bacterial spoilage in fish, were identified. These findings, supported by examination of histological sections of Anisakis -positive samples, along with the increased bacterial load in the flesh indicate that certain shelf life reducing SSB are introduced into the fish flesh by A. simplex larvae.

SUMMARYA multi-marker nuclear genotyping approach was performed on larval and adult specimens ofAnisakisspp. (N= 689) collected from fish and cetaceans in allopatric and sympatric areas of the two speciesAnisakis pegreffiiandAnisakis simplex(s. s.), in order to: (1) identify specimens belonging to the parental taxa by using nuclear markers (allozymes loci) and sequence analysis of a new diagnostic nuclear DNA locus (i.e. partial sequence of the EF1α−1nDNA region) and (2) recognize hybrid categories. According to the Bayesian clustering algorithms, based on those markers, most of the individuals (N= 678) were identified as the parental species [i.e.A. pegreffiiorA. simplex(s. s.)], whereas a smaller portion (N= 11) were recognized as F1hybrids. Discordant results were obtained when using the polymerase chain reaction–restriction fragment length polymorphisms (PCR–RFLPs) of the internal transcribed spacer (ITS) ribosomal DNA (rDNA) on the same specimens, which indicated the occurrence of a large number of ‘hybrids’ both in sympatry and allopatry. These findings raise the question of possible misidentification of specimens belonging to the two parentalAnisakisand their hybrid categories derived from the application of that single marker (i.e. PCR–RFLPs analysis of the ITS of rDNA). Finally, Bayesian clustering, using allozymes and EF1α−1nDNA markers, has demonstrated that hybridization betweenA. pegreffiiandA. simplex(s. s.) is a contemporary phenomenon in sympatric areas, while no introgressive hybridization takes place between the two species.

Grey gurnard ( Eutrigla gurnardus L.) is an abundant and widely distributed demersal fish in the North Sea. It is currently considered a sustainable stock, thus permitting future increased utilisation as a food resource. In order to address both consumer safety and aesthetical product quality, the occurrence and distribution of anisakid nematode larvae in Grey gurnard from two localities in the North Sea were investigated. Two anisakid species were recorded, i.e. Hysterothylacium aduncum in the viscera, and Anisakis simplex in both the viscera and flesh of the fish. Virtually all gurnards were infected with nematode larvae. However, H. aduncum was significantly less abundant than A. simplex in the fish from both localities. Only for the gurnards from the northernmost sampling locality there was a significantly positive correlation between host body weight and total A. simplex abundance. No such correlation was found for H. aduncum in either locality. Separate analyses of the Anisakis infection data in gurnards of marketable size (≥250 g) and pooled for both localities, revealed 83% prevalence of A. simplex larvae in the fish flesh, ranging 1–16 in intensity. The relative larval distribution between the viscera and flesh was 89 and 11%, respectively. Moreover, a significantly positive correlation was found between A. simplex occurring in the viscera and the flesh of this particular host size group, i.e. the number of larvae in the flesh appeared to increase with increasing infection level in the viscera. In general, Grey gurnard from the North Sea can be considered as heavily infected with nematode larvae. Especially the comparatively high abundance of A. simplex larvae in the flesh is of concern regarding the possible intensified utilization of Grey gurnard as a food resource.

There is a growing awareness by consumers and food safety authorities regarding the possible presence of parasites or parasite-related potentially hazardous substances in seafood. Anisakis simplex is among the most frequently occurring parasites in wild-caught marine fish. Except for various visual inspection techniques and PCR-based methods for the detection of more or less intact worms or parasite DNA, respectively, there are at present no validated methods for the quantification of A. simplex proteins in processed fish products. This work describes the development and validation of a sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification and analysis of proteins from A. simplex in seafood products. The ELISA is based on a polyclonal rabbit anti-A. simplex antibody for capture and a biotinylated conjugate of the same antibody for detection. The ELISA is specific for A. simplex and does not cross-react with other species. Recoveries ranged from 72–101% in typical food matrixes, while intra- and inter-assay precisions were

The carry-over of certain feed components into animal products can be of concern for human health. The safety assessment of chemical contaminants including natural toxins, agrochemicals, veterinary drugs, and environmental pollutants is a key element of the “farm-to-fork” (“One Health”) approach. The transmissibility of proteinaceous feed constituents such as enzymes, proteins from genetically engineered crops, and infectious prions in animal meal has also become of interest; but the transfer of proteins with allergic potential is little studied. In the present study, an exploratory zebrafish feeding trial using feed containing 20 percent of processed larvae of the marine fish parasite Anisakis simplex was performed as a proof-of-principle experiment. After a 2-week exposure period, anisakid peptides were detected in zebrafish tissue by high-resolution liquid-chromatography Orbitrap mass spectrometry and immunostaining using specific polyclonal antibodies or sera from patients with confirmed allergy to A. simplex. Since fishmeal produced from marine pelagic fish is an important feed component in the culture of Atlantic salmon and in the poultry industry, it should be considered as a source of potentially allergenic peptides in the final products. Furthermore, the substitution of fishmeal with plant proteins would not eliminate the potential health risk by allergen carry-over since crops of high nutritional value such as legumes also contain important food allergens. If our preliminary results from the present zebrafish feeding trial should be confirmed in necessary follow-up experiments, the question of labeling information on fish and animal food products raised on feed containing potentially allergenic ingredients could arise in order to minimize the exposure risk of allergic consumers. acceptedVersion

A new species, Myxobolus balantiocheili, is described from the central nervous system of wild and cultured tricolor sharkminnow Balantiocheilos melanopterus from Thailand. The infection was associated with emaciation and severe neurological symptoms, including sudden uncoordinated darting, rolling and pitching. Whitish cysts of varying shape, measuring 70 to 500 microm, were located in the brain, spinal cord and in the meninx, and were sometimes accompanied by haemorrhages. In the brain, most cysts were located in the medulla oblongata and the mesencephalon. Cysts with a segmental distribution were observed in the meninx of the spinal cord. Single di- to hexasporic pansporoblasts, and free mature spores, were frequently seen in both the white and grey matter of the brain and spinal cord, mostly associated with blood vessels. Mature spores varied in shape from oval to ellipsoid, measuring 12.3 +/- 0.59 (range 10.6 to 13.3) x 10.0 +/- 0.47 (9.2 to 10.9) x 6.9 +/- 0.20 (6.7 to 7.1) microm. Spore size and morphology, in addition to fish-host species and apparently narrow tissue tropism, clearly distinguishes this new Myxobolus. No marked inflammatory response or gliosis was associated with the lesions, but compression and degeneration of the nervous tissue surrounding large cysts was observed. We suggest that the high temperature of the fish hosts' tropical habitat may have increased the rate of development and the virulence of M. balantiocheili.

The freshwater fish nematode Camallanus cotti Fujita, 1927 (Spirurida: Camallanidae) is naturally distributed in East, South and Southeast Asia. During the past three or four decades the species has been disseminated to Europe, North America, Australia and Hawaii, mainly due to the extensive ornamental fish trade. In the present study, its development and larval morphogenesis is described. Newborn first-stage larvae are ingested by copepods and within hours reach the haemocoel, where the worms moult twice. At 22 degrees C ambient temperature, fully-developed third-stage larvae appear on day 11 p.i. After transmission into the fish intestine two further moults occur before the adult stage is reached. At 23 degrees C water temperature, the final moult is initiated around day 33 p.i. in males and between 34 and 42 days p.i. in females. Morphologically the young and older adults are similar, but the buccal capsule of both young males and young females is not yet fully sclerotised. Based on the close similarity in the development and adult morphology of the buccal capsule in C. cotti and three congeners from Europe and North America, we suggest, in general agreement with earlier workers, that the Camallanus RaillietHenry, 1915 originated in tropical Asia and subsequently radiated to species when adapting to new climatic and/or host-related conditions in the Old World and the New World. C. cotti may thus represent a lineage of speciation which did not apparently involve extensive changes in relation to their natural geographical distribution or definitive host range.

The parasitic fish nematode Camallanus cotti has been reported from a number of freshwater fish species around the world. Its wide geographical distribution seems mainly to be the result of anthropogenic dissemination due to extensive ornamental fish trade. In most reports it is assumed that C. cotti's life cycle involves cyclopoid copepods as intermediate host and various freshwater fishes as final host. However, the species' relatively frequent and persistent occurrence in aquaria worldwide strongly indicates flexibility in its life cycle, i.e. the ability to infect the final host directly. The present study has shown that under aquaria conditions, without any presence of copepods, C. cotti is able to infect various phylogenetically distant fish species directly for at least three generations. It was further shown that the infective free-living first-stage larvae may survive for more than three weeks in the host-external environment and that their host-attracting behaviour is not precluding direct transmission to the final fish host. Any treatment for C. cotti under aquaculture or aquarium conditions should be directed towards both individual infected fish hosts as well as the free-living larvae on the substrate.

The third stage larvae of the parasitic nematode Anisakis simplex commonly occur in most commercially important fish species of the North Atlantic, including Norwegian spring spawning herring (Clupea harengus L.). The presence of nematode larvae in the flesh of fish may significantly lower the aesthetical quality of the product, or even pose a consumer health risk, especially with regard to the possible allergenic nature of the larvae or molecular traces thereof. In this study, the occurrence and spatial distribution of A. simplex larvae in comparable size groups of Norwegian spring spawning herring caught in the north-eastern Norwegian Sea in October 2004 and in the outer basin of Vestfjorden, northern Norway, in November 2007, was investigated. Emphasis was put on manually- and industrially produced, i.e. automatically trimmed and skinned fillets of herring. The overall larval prevalence was 98-100% in the herring of all size groups and the abundance increased with increasing body weight in both sampling years. On an average 3.5% of the larvae were found in the belly flaps, i.e. the ventral portion of the body musculature covering the visceral cavity on both sides, while 0.5% occurred in the dorsal part of the fillets. The larval prevalence varied from 42 to 70% and 8 to 10% in the manually- and industrially produced fillets, respectively. Thus, any product that is based on industrially produced fillets of Norwegian spring spawning herring may still carry nematode larvae when put on the market. However, compared to the manually produced ones, especially those untrimmed, the probability of A. simplex larvae to be present in industrially produced fillets appears to be approximately 5-8 times lower.

Three Haliotrema spp. are reported from the Vietnamese grouper. Morphological and morphometric characters show minor deviations from original descriptions of H. cromileptis Young, 1968 and H. epinepheli Young, 1968 . The third encountered species ( Haliotrema sp.) appears to be new to science. Genetically, H. cromileptis clusters with Bravohollisia , Pseudohaliotrema , and Haliotrema . The group is well supported by partial large subunit rDNA (LSU), complete small subunit rDNA (SSU), and partial SSU + ITS1 rDNA analyses. Ingroup phylogenetic relationships are not well resolved. Haliotrema cromileptis , H. fleti Young, 1968 , and Pseudohaliotrema sphincteroporus Yamaguti, 1953 are closely related to a monophyletic group of 5 Haliotrema spp. characterized by bell- or horn-shaped bases of the male copulatory organ (MCO), which contains an accessory piece. Based on SSU rDNA, H. cromileptis is a sister species to P. sphincteroporus and, together, they form a clade to 3 other Haliotrema spp. characterized by a bell-shape based MCO with an accessory piece. Data analysis conducted on partial SSU + ITS1 rDNA confirms the close phylogenetic relationship of H. cromileptis , H. fleti , H. chenhsintaoi Zhang, 2001 (possessing a horn-shaped base of the MCO), and Bravohollisia rosetta Lin, 1995. However, because major differences in diagnostic characters exist, this genetic relationship needs further elucidation.

Publisher Summary Parasitism is a highly successful way of life as it has evolved independently in almost every phylum of protists, animals and plants. However, the degree to which parasites might affect their hosts is highly variable, ranging from an apparently uncomplicated co-existence with low virulence to situations where they might strongly affect entire host populations, under both natural or aquaculture conditions. In many parasite groups, but especially among aquatic ones, there are intricate and complex life cycles. These generally involve one or more intermediate hosts where larval development or asexual multiplication occurs and a definitive host in which to breed and from which to shed their offspring in order to ensure dispersal. Besides the species of concern to seafood safety, other frequently occurring parasites affect the food quality as they might severely reduce the aesthetic appearance of the product. Thus, a number of different fish parasites might have severe economic impact, either related to their pathogenicity, often resulting in substantial losses under aquaculture conditions, or due to their importance regarding the quality and safety of fish and fishery products. The continuously growing awareness by consumers and public food authorities as to the possible presence of parasites or parasite-related quality defects in seafood emphasizes the importance of providing the fish processing industry with up-to-date knowledge on the occurrence, detection, and control regarding the most important parasite species of fish.

Members of the myxosporean genus Kudoa occur in various marine teleosts worldwide. Several species are of concern to the fishery and aquaculture industries as they may produce unsightly cysts in the fish host's musculature or are associated with postmortem myoliquefaction of the fish muscle, commonly referred to as 'soft flesh'. This study describes the occurrence and effects on a host of a Kudoa species in Atlantic mackerel, Scomber scombrus, from the northern North Sea. Generalized postmortem myoliquefaction associated with Kudoa sp. occurred in 0.8% of the examined fish, i.e. 11 of 1339 mackerel developed 'soft flesh'. There was a significant difference in the prevalence of myoliquefaction between medium sized (400-600 g) and large mackerel (>600 g). The prevalence reached 8.9% in the latter host size group. No subclinical infections of Kudoa sp. were detected when examining fresh muscle (n = 103) and blood (n = 165) samples for spores using light microscopy. Affected mackerel developed generalized myoliquefaction after 38-56 h post-catch. No inflammatory host response was associated with the presence of plasmodia within single body muscle fibres of 'soft flesh' affected fish. Based on comparison of myxospore dimensions and analysis of the nuclear small subunit (SSU) ribosomal DNA, the present Kudoa species is assigned to Kudoa thyrsites. However, due to the species' apparently very wide geographical distribution and host range, its varying effect on different fish host species, together with the still unknown life cycle of Kudoa spp., the taxonomic status of K. thyrsites appears not to be fully resolved.

The third-stage larvae of the parasitic nematode Anisakis simplex commonly occur in most commercially important fish species from the North Atlantic. In this study, the occurrence and site distribution of Anisakis larvae in various size groups of Norwegian spring spawning herring (Clupea harengus), blue whiting (Micromesistius poutassou), and mackerel (Scomber scombrus) from the Northeast Atlantic was investigated. Although most of the larvae were found in the abdominal cavity, the small proportion lodged in the flesh was sufficient to affect food quality and safety. However, the prevalence of Anisakis larvae in fish fillets varied considerably among the size groups of each species: 15 to 60% in herring, 32 to 77% in mackerel, and 89 to 100% in blue whiting. The commonly recommended nematode detection method in the fish processing industry is candling, i.e., a brief visual inspection on a light table. Comparison of successively more accurate detection methods, i.e., candling, enzymatic degradation, and UV illumination, for fillets of each fish species revealed that only 7 to 10% of the nematode larvae present in the fillets were detected by candling. The candling efficiency was apparently independent of fillet thickness. Thus, candling is not sufficient to detect the majority of the nematodes that are actually present in the fillets of pelagic fish from the Northeast Atlantic. These findings emphasize the importance of adequate deep-freezing of any fresh pelagic fish product, especially if it is intended for consumption in a raw or semiraw state.